Journal: European journal of medicinal chemistry

Article Title: Design, synthesis, and biological evaluation of a series of new anthraquinone derivatives as anti-ZIKV agents.

doi: 10.1016/j.ejmech.2023.115620

Figure Lengend Snippet: Fig. 7. Compound 22 decreases ZIKV NS5 RdRp ac tivities. The effect of 22 on ZIKV NS5 activity. HEK293T cells were transfected with Gluc reporter plasmid or co- transfected Gluc reporter and ZIKV NS5 plasmid and then treated with different concentrations (1, 5 and 10 μM) of 22 for 24h. Gluc activity in supernatants was quantified. 20 μM of fidaxomicin was treated as a positive control which is a potent inhibitor of ZIKV. 1 % DMSO was treated as a negative control. Western Blot analysis of the effect of the expression level of NS5 (upper panel). GAPDH was used as a loading control (lower panel). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: A potent ZIKV NS5 inhibitor fidaxomicin (MedChemExpress LLC, Shanghai, China) at a concentration of 20 μM was used as the positive control.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Positive Control, Negative Control, Western Blot, Expressing, Control

Journal: European journal of medicinal chemistry

Article Title: Design, synthesis, and biological evaluation of a series of new anthraquinone derivatives as anti-ZIKV agents.

doi: 10.1016/j.ejmech.2023.115620

Figure Lengend Snippet: Fig. 6. 22 binds directly to the ZIKV NS5 protein. (A) The overview of ZIKV NS5 bound to 22 and 22 shown as green sticks. (B) Two-dimensional ligand- interaction maps of co-crystals of ZIKV NS5 domains bound with 22 were generated using Molecular Operating Environment. Polar residues are colored light purple, charged residues have an additional blue ring, and lipophilic residues are green. The degree of solvent exposure is shown by the blue halos. H-bond interactions to the amino acid side chain or main chain are shown as dashed green arrows, respectively, pointing towards the H-bond acceptor. (C) and (D) SPR assay to examine and characterize the binding of 22 or ribavirin to ZIKV RdRp, using a BIAcore T100 system. ZIKV RdRp protein was immobilized on a CM5 chip, respectively. The analytes were consisting of serial dilutions of 22 or ribavirin, as indicated. The Kd values presented in (C) and (D) were calculated via the BIAcore T100 analysis software (BIAevaluation Version 3.1).

Article Snippet: A potent ZIKV NS5 inhibitor fidaxomicin (MedChemExpress LLC, Shanghai, China) at a concentration of 20 μM was used as the positive control.

Techniques: Generated, Solvent, SPR Assay, Binding Assay, Software

Journal: Frontiers in Chemistry

Article Title: Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3 H )-ones targeting the ZIKV NS5 RNA dependent RNA polymerase

doi: 10.3389/fchem.2022.1010547

Figure Lengend Snippet: The interaction between 4w and ZIKV NS5 protein. (A) WB detection of ZIKV NS5 protein expression under different temperature gradients after 4w treatment; (B) NS5 protein grayscale analysis and ZIKV NS5 protein expression after 4w treatment observed changes in aggregation temperature; data is the mean (±SD) of three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with ZIKV E (1:2,000) and NS5 antibody (1:2,000) overnight at 4°C (Sino Biological, China) then add the corresponding secondary antibody and incubate at room temperature for 2 h. A specific signal was presented with a chemiluminescent substrate.

Techniques: Expressing

Journal: Frontiers in Chemistry

Article Title: Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3 H )-ones targeting the ZIKV NS5 RNA dependent RNA polymerase

doi: 10.3389/fchem.2022.1010547

Figure Lengend Snippet: Compound 4w inhibited the expression of ZIKV E and NS5 protein. (A) Western blot detected the inhibitory effect of 4w on ZIKV E and NS5 protein under the concentration gradient of 4w ; (B) Grayscale analysis and statistics of the inhibition of 4w on ZIKV E and NS5 protein under the concentration gradient of WB detection; Data is the mean (±SD) of three experiments, with DMSO as a positive contro * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (C) Detected the inhibitory effect of 4w on ZIKV E protein by immunofluorescence.

Article Snippet: Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with ZIKV E (1:2,000) and NS5 antibody (1:2,000) overnight at 4°C (Sino Biological, China) then add the corresponding secondary antibody and incubate at room temperature for 2 h. A specific signal was presented with a chemiluminescent substrate.

Techniques: Expressing, Western Blot, Concentration Assay, Inhibition, Immunofluorescence

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet: (A–D) 293T cell pools stably expressing Cas9 nuclease and an sgRNA targeting EMC6 were subsequently transiently co-transfected with constructs encoding HA-tagged DENV non-structural proteins and NS1-FLAG (A, C, and D) or GFP (B) as a transfection control. (E) ImageJ (NIH) was used to quantify band intensities for western blots of each of the DENV non-structural proteins in EMC6 knockout cells compared with wild-type from (A)–(D). Each dot represents a biological replicate. Bars represent mean ± SD. The Mann-Whitney U test was performed to assess statistical significance (*p < 0.05 and **p < 0.005 compared to NS1). (F–K) HEK293 cells were transfected with siRNAs against EMC1 or a scrambled negative control (scr). Forty-eight hours later, cells were transfected with a replication-defective full-length ZIKV cDNA (F and G) or with plasmids encoding individual S-tagged ZIKV non-structural proteins NS5 (H), NS4A (I), NS4B (J), or NS2B (K). The asterisk in (G) indicates a nonspecific background band. For (A)–(D) and (F)–(K) 24 h post-transfection, cells were lysed and proteins were separated using SDS-PAGE followed by western blotting for the indicated proteins. Each blot is representative of a minimum of two biological replicates. See also .

Article Snippet: Anti-ZIKV NS5 , Genetex , Cat# GTX133312; RRID: AB_2750559.

Techniques: Stable Transfection, Expressing, Transfection, Construct, Control, Western Blot, Knock-Out, MANN-WHITNEY, Negative Control, SDS Page

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet: (A) The TMHMM version 2.0 algorithm was used to predict the presence of transmembrane helices in 2k-NS4B of DENV (UniProtKB: P29990). This plot depicts the probability of a given residue being located within a transmembrane helix, where greater values reflect greater transmembrane probability. The indicated amino acid positions on the x axis indicate the boundaries of each predicted transmembrane domain with scores above 0.3 for each predicted transmembrane domain. (B) HEK293T cells stably expressing Cas9 and sgRNA targeting EMC6 were co-transfected to express the indicated C-terminally GFP-tagged NS4B mutants and NS1-FLAG as a transfection control. 2k-96 indicates a C-terminal truncation at amino acid 97. 2k-58 indicates a C-terminal truncation at amino acid 59. Δ32–96 indicates a deletion from 32 to 96. Twenty-four hours post-transfection, cells were lysed and proteins were resolved using SDS-PAGE followed by western blotting for the indicated proteins. (C) Band intensitiesforNS4Bwild-type and Δ32–96 in EMC6 knockout cells compared with wild-type cells. Each point represents a biological replicate from cells transfected with NS4B-GFP (circles) or NS4B-HA (triangles). (D) Schematic of NS4B mutations to alter hydrophobicity of the pTM1 and pTM2 helices. Both of these helices are speculated to be membrane associated, with red representing hydrophobic and blue representing charged and polar hydrophilic residues, respectively (middle). Mutagenesis of the hydrophobic residues to lysine (top) is expected to result in decreased membrane association of pTM1 and pTM2. Conversely, mutagenesis of charged or polar residues to leucine (bottom) is expected to result in the insertion of pTM1 and pTM2 into the membrane. (E and F) Wild-type 293T cells or cells stably expressing Cas9 and sgRNA targeting EMC6 were transfected to express the indicated NS4B-HA mutants with substitutions to lysine residues (less hydrophobic) shown in (E) and substitutions to leucine residues (more hydrophobic) shown in (F). Twenty-four hours post-transfection, cells were lysed and proteins were resolved using SDS-PAGE followed by western blotting for the indicated proteins. Blots are representative of a minimum of three independent experiments.(G) Bands were quantitated using ImageJ and represented as a ratio of band intensity in EMC6 knockout versus wild-type cells. Each point represents a biological replicate, with bars representing mean ± SD. Triangles represent quantification from blots of NS4B with PNGase treatment, while circles represent quantification from blots of NS4B without PNGase treatment. Statistical significance was assessed using Dunnett’s test for multiple comparisons (**p < 0.005 and ***p < 0.0005 compared with WT NS4B). (H) Model of EMC interaction with theSec61 translocon and the flavivirus protein. Depicted is a flavivirus genomic RNA (upper left) being translated by a ribosome (blue) at the cytosolic face of the ER. Transmembrane domains are cotranslationally inserted into the ER membrane by the Sec61 translocon (brown). The EMC (yellow) is associated with the translocon and assists in the proper insertion and stabilization of certain multi-pass transmembrane domain proteins at the time of protein translation and/or translocation. The expression of both NS4A and NS4B of DENV and ZIKV is dependent on the EMC. Expression of the next protein in the flavivirus polypeptide, NS5, is also decreased in EMC-deficient cells when NS5 is expressed on the same polyprotein as NS4A and NS4B but not when expressed by itself.

Article Snippet: Anti-ZIKV NS5 , Genetex , Cat# GTX133312; RRID: AB_2750559.

Techniques: Residue, Stable Transfection, Expressing, Transfection, Control, SDS Page, Western Blot, Knock-Out, Membrane, Mutagenesis, Translocation Assay

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet:

Article Snippet: Anti-ZIKV NS5 , Genetex , Cat# GTX133312; RRID: AB_2750559.

Techniques: Recombinant, Protease Inhibitor, Membrane, Luciferase, Transfection, Virus, Software